Journal Article

Isolation and Partial Characterization of the NADH Dehydrogenase Complex from Barley Chloroplast Thylakoids

María José Quiles, María Eugenia Albacete, Bartolomé Sabater and Juan Cuello

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 37, issue 8, pages 1134-1142
Published in print December 1996 | ISSN: 0032-0781
e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/oxfordjournals.pcp.a029064
Isolation and Partial Characterization of the NADH Dehydrogenase Complex from Barley Chloroplast Thylakoids

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  • Molecular and Cell Biology
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Using chloroplasts from barley leaves we attempt to purify and partially characterize the NADH dehydrogenase complex. The enzymatic activity was assayed as NADH-ferricyanide and NADH-nitro blue tetrazolium oxidoreductase. Analyzed by SDS-polyacrylamide gel electrophoresis and subsequent enzymatic renaturation, the chloroplast soluble fraction contains principally a 66 kDa enzyme. The membranous fraction solubilized with deoxycholate and analyzed by native electrophoresis and NADH-nitro blue tetrazolium staining revealed three enzymes: one with similar electrophoretic mobility to that described for the soluble enzyme, another one which is a complex separated in 3% polyacrylamide gel and a third one, another complex separated in the top of the 5–22% linear polyacrylamide gel gradient. The complex polypeptidic patterns were similar but different to those found for any thylakoidal proteinic complex known. Nine major polypeptides were detected in the complex polypeptidic patterns, four of them constituents of the small size thylakoid enzyme. The molecular masses of six polypeptides agreed with those indicated as encoded by 6 chloroplast ndh genes. All the enzymes, including the 66 kDa soluble enzyme, contained a 53 kDa polypeptide, which is probably the NADH-binding complex subunit. Isoelectric focusing of the thylakoidal enzyme points to a basic isoelectric point. Ion-exchange or hydroxylapatite column chromatography followed by native electrophoresis of the active fractions only separated the small size enzyme, which showed complex inactivation.

Keywords: Barley (Hordeum vulgare); Chloroplasts; Enzymatic purification; NADH dehydrogenase complex; Polypeptide analysis; Thylakoids

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Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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